Disodium salts, monohydrates, and ethanol solvates for delivering active agents

ABSTRACT

The inventors have discovered that the disodium salt of certain delivery agents has surprisingly greater efficacy for delivering active agents than the corresponding monosodium salt. Furthermore, the inventors have discovered that the disodium salts of these delivery agents form solvates with ethanol and hydrates with water. The delivery agents have the formula 
     
       
         
         
             
             
         
       
     
     wherein
         R 1 , R 2 , R 3 , and R 4  are independently hydrogen, halogen, C 1 -C 4  alkyl, or C 1 -C 4  alkoxy; and   R 5  is a substituted or unsubstituted C 2 -C 16 alkylene, substituted or unsubstituted C 2 -C 16  alkenylene, substituted or unsubstituted C 1 -C 12  alkyl(arylene), or substituted or unsubstituted aryl(C 1 -C 12  alkylene). The hydrates and solvates of present invention also have surprisingly greater efficacy for delivering active agents, such as heparin and calcitonin, than their corresponding monosodium salts and free acids. The present invention provides an alcohol solvate, such as ethanol solvate, of a disodium salt of a delivery agent of the formula above. The invention also provides a hydrate of a disodium salt of a delivery agent of the formula above. Preferred delivery agents include, but are not limited to, N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), N-(10-[2-hydroxybenzoyl]amino)decanoic acid (SNAD), and sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC). The invention also provides methods of preparing the disodium salt, ethanol solvate, and hydrate and compositions containing the disodium salt, ethanol solvate, and/or hydrate.

This application claims the benefit of U.S. patent application Ser. No.60/127,754, filed Apr. 5, 1999; U.S. patent application Ser. No.60/186,143, filed Mar. 1, 2000; U.S. patent application Ser. No.60/186,142, filed Mar. 1, 2000; and U.S. patent application Ser. No.60/191,286, filed Mar. 21, 2000.

FIELD OF THE INVENTION

The present invention relates to a disodium salt of a delivery agent,such as N-(5-chlorosalicyloyl)-8-aminocaprylic acid,N-(10-[2-hydroxybenzoyl]amino)decanoic acid, orN-(8-[2-hydroxybenzoyl]amino)caprylic acid, an ethanol solvate of thedisodium salt, and a monohydrate of the disodium salt for deliveringactive agents and methods of preparing the same.

BACKGROUND OF THE INVENTION

U.S. Pat. Nos. 5,773,647 and 5,866,536 disclose compositions for theoral delivery of active agents, such as heparin and calcitonin, withmodified amino acids, such as N-(5-chlorosalicyloyl)-8-aminocaprylicacid (5-CNAC), N-(10-[2-hydroxybenzoyl]amino)decanoic acid (SNAD), andN-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC). Many currentcommercial formulations containing an active agent, such as heparin andcalcitonin, are delivered by routes other than the oral route.Formulations delivered orally are typically easier to administer than byother routes and improve patient compliance.

There is a need for improved pharmaceutical formulations for orallyadministering active agents, such as heparin and calcitonin.

SUMMARY OF THE INVENTION

The inventors have discovered that the disodium salt of certain deliveryagents has surprisingly greater efficacy for delivering active agentsthan the corresponding monosodium salt. Furthermore, the inventors havediscovered that the disodium salts of these delivery agents formsolvates with ethanol and hydrates with water. The delivery agents havethe formula

wherein

R¹, R², R³, and R⁴ are independently hydrogen, —OH, —NR⁶R⁷, halogen,C₁-C₄ alkyl, or C₁-C₄ alkoxy;

R⁵ is a substituted or unsubstituted C₂-C₁₆ alkylene, substituted orunsubstituted C₂-C₁₆ alkenylene, substituted or unsubstituted C₁-C₁₂alkyl(arylene), or substituted or unsubstituted aryl(C₁-C₁₂ alkylene);and

R⁶ and R⁷ are independently hydrogen, oxygen, or C₁-C₄ alkyl. Thehydrates and solvates of the present invention also have surprisinglygreater efficacy for delivering active agents, such as heparin andcalcitonin, than their corresponding monosodium salts and free acids.

The present invention provides an alcohol solvate, such as methanol,ethanol, propanol, propylene glycol, and other hydroxylic solvates, of adisodium salt of a delivery agent of the formula above. According to onepreferred embodiment, the alcohol solvate is ethanol solvate. Theinvention also provides a hydrate, such as a monohydrate, of a disodiumsalt of a delivery agent of the formula above. Preferred delivery agentsinclude, but are not limited to, N-(5-chlorosalicyloyl)-8-aminocaprylicacid (5-CNAC), N-(10-[2-hydroxybenzoyl]amino)decanoic acid (SNAD),N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC),8-(N-2-hydroxy-4-methoxybenzoyl)aminocaprylic acid (as shown as compound67 in U.S. Pat. No. 5,773,647), and N-(9-(2-hydroxybenzoyl)aminononanicacid (or 9-salicyloylaminononanoic acid) (as shown as compound 35 inU.S. Pat. No. 5,773,647).

The present invention also provides a method of preparing the disodiumsalt of the present invention by drying the ethanol solvate of thepresent invention. According to a preferred embodiment, the ethanolsolvate is prepared by the method described below.

Another embodiment of the invention is a method of preparing the ethanolsolvate of the present invention. The method comprises dissolving adelivery agent of the formula above in ethanol to form a deliveryagent/ethanol solution; (b) reacting the delivery agent/ethanol solutionwith a molar excess of a sodium containing salt to form the ethanolsolvate.

Yet another embodiment of the invention is a method of preparing thehydrate of the present invention. The method comprises (a) obtaining anethanol solvate of the disodium salt of the delivery agent; (b) dryingthe solvate to form an anhydrous disodium salt; and (c) hydrating theanhydrous disodium salt to form the hydrate.

Yet another embodiment of the present invention is a compositioncomprising a disodium salt of the delivery agent.

Yet another embodiment of the invention is a composition comprising atleast one disodium salt, ethanol solvate, or hydrate of the presentinvention and at least one active agent. Preferred active agentsinclude, but are not limited to, heparin and calcitonin. The compositionmay be formulated into a dosage unit form, such as an oral dosage unitform.

Yet another embodiment of the present invention is a method foradministering an active agent to an animal in need thereof comprisingadministering to the animal the composition of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The term “substituted” as used herein includes, but is not limited to,substitution with any one or any combination of the followingsubstituents: halogens, hydroxide, C₁-C₄ alkyl, and C₁-C₄ alkoxy.

The terms “alkyl”, “alkoxy”, “alkylene”, “alkenylene”, “alkyl(arylene)”,and “aryl(alkylene)” include, but are not limited to, linear andbranched alkyl, alkoxy, alkylene, alkenylene, alkyl(arylene), andaryl(alkylene) groups, respectively.

Disodium Salt

The disodium salt may be prepared from the ethanol solvate byevaporating or drying the ethanol by methods known in the art to formthe anhydrous disodium salt. Generally, drying is performed at atemperature of from about 80 to about 120, preferably from about 85 toabout 90, and most preferably at about 85° C. Typically, the drying stepis performed at a pressure of 26″ Hg or greater. The anhydrous disodiumsalt generally contains less than about 5% by weight of ethanol andpreferably less than about 2% by weight of ethanol, based upon 100%total weight of anhydrous disodium salt.

The disodium salt of the delivery agent may also be prepared by making aslurry of the delivery agent in water and adding two molar equivalentsof aqueous sodium hydroxide, sodium alkoxide, or the like. Suitablesodium alkoxides include, but are not limited to, sodium methoxide,sodium ethoxide, and combinations thereof.

Yet another method of preparing the disodium salt is by reacting thedelivery agent with one molar equivalent of sodium hydroxide to form amonosodium salt of the delivery agent and then adding an additional onemolar equivalent of sodium hydroxide to yield the disodium salt.

The disodium salt can be isolated as a solid by concentrating thesolution containing the disodium salt to a thick paste by vacuumdistillation. This paste may be dried in a vacuum oven to obtain thedisodium salt of the delivery agent as a solid. The solid can also beisolated by spray drying an aqueous solution of the disodium salt.

The delivery agent may be prepared by methods known in the art, such asthose described in U.S. Pat. Nos. 5,773,647 and 5,866,536, respectively.

Another aspect of the invention is a composition comprising at leastabout 20% by weight and preferably at least about 60% by weight of thedisodium salt of the delivery agent, based upon 100% total weight of thedelivery agent and salts thereof in the composition. According to oneembodiment, the composition comprises at least about 10, 30, 40, 50, 70,or 80% by weight of the disodium salt of the delivery agent, based upon100% total weight of the delivery agent and salts thereof in thecomposition. More preferably, the composition comprises at least about90% by weight of the disodium salt of the delivery agent, based upon100% total weight of the delivery agent and salts thereof in thecomposition.

Most preferably, the composition comprises substantially pure disodiumsalt of the delivery agent. The term “substantially pure” as used hereinmeans that less than about 4% and preferably less than about 2% byweight of the delivery agent in the composition is not a disodium salt,based upon 100% total weight of the delivery agent and salts thereof inthe composition.

Ethanol Solvate

The term “ethanol solvate” as used herein includes, but is not limitedto, a molecular or ionic complex of molecules or ions of ethanol solventwith molecules or ions of the disodium salt of the delivery agent.Typically, the ethanol solvate contains about one ethanol molecule orion for every molecule of disodium salt of the delivery agent.

The ethanol solvate of the disodium salt of the delivery agent may beprepared as follows. The delivery agent is dissolved in ethanol.Typically, each gram of delivery agent is dissolved in from about 1 toabout 50 mL of ethanol and preferably from about 2 to about 10 mL ofethanol. The delivery agent/ethanol solution is then reacted with amolar excess of a sodium containing salt, such as a monosodiumcontaining salt, relative to the delivery agent, i.e., for every mole ofdelivery agent there is more than one mole of sodium cations. Thisreaction yields the ethanol solvate. Suitable monosodium containingsalts include, but are not limited to, sodium hydroxide; sodiumalkoxides, such as sodium methoxide and sodium ethoxide; and anycombination of any of the foregoing. Preferably, at least about twomolar equivalents of the monosodium containing salt are added to theethanol solution, i.e., for every mole of delivery agent there is atleast about two moles of sodium cations. Generally, the reaction isperformed at a temperature at or below the reflux temperature of themixture, such as at ambient temperature.

The ethanol solvate may then be recovered by methods known in the art.For example, the slurry resulting from the addition of sodium hydroxideto the delivery agent/ethanol solution may be concentrated byatmospheric distillation. The concentrated slurry may then be cooled andthe solid product recovered by filtration. The filter cake, i.e., thefiltrate, may be vacuum dried to obtain the ethanol solvate.

Hydrate

The term “hydrate” as used herein includes, but is not limited to, (i) asubstance containing water combined in the molecular form and (ii) acrystalline substance containing one or more molecules of water ofcrystallization or a crystalline material containing free water.Compositions containing the hydrate of the disodium salt preferablycontain at least about 80%, more preferably at least about 90%, and mostpreferably about 95% by weight of the monohydrate of the dissodium salt,based upon 100% total weight of hydrate of disodium salt in thecomposition. According to a preferred embodiment, the compositioncontains at least about 98% by weight of the monohydrate of thedissodium salt, based upon 100% total weight of hydrate of disodium saltin the composition.

The hydrate may be prepared by drying the ethanol solvate to form ananhydrous disodium salt as described above and hydrating the anhydrousdisodium salt. Preferably, the monohydrate of the disodium salt isformed. Since the anhydrous disodium salt is very hygroscopic, thehydrate forms upon exposure to atmospheric moisture. Generally, thehydrating step is performed at from about ambient temperature to about50° C. and in an environment having at least about 50% relativehumidity. Preferably, the hydrating step is performed at from aboutambient temperature to about 30° C. For example, the hydrating step maybe performed at 40° C. and 75% relative humidity. Alternatively, theanhydrous disodium salt may be hydrated with steam.

According to one preferred embodiment, the drying and hydrating stepsare performed in an oven. Preferably, the material is not exposed to theatmosphere until both steps are complete.

Disodium Salt, Ethanol Solvate, and Hydrate Compositions and Dosage UnitForms

The invention also provides a composition, such as a pharmaceuticalcomposition, comprising at least one of a disodium salt, ethanolsolvate, or hydrate of the present invention and at least one activeagent. The composition of the present invention typically contains adelivery effective amount of one or more disodium salts, ethanolsolvates, and/or hydrates of the present invention, i.e., an amount ofthe disodium salt, ethanol solvate, and/or hydrate sufficient to deliverthe active agent for the desired effect.

Active agents suitable for use in the present invention includebiologically active agents and chemically active agents, including, butnot limited to, pesticides, pharmacological agents, and therapeuticagents.

For example, biologically or chemically active agents suitable for usein the present invention include, but are not limited to, proteins;polypeptides; peptides; hormones; polysaccharides, and particularlymixtures of muco-polysaccharides; carbohydrates; lipids; other organiccompounds; and particularly compounds which by themselves do not pass(or which pass only a fraction of the administered dose) through thegastro-intestinal mucosa and/or are susceptible to chemical cleavage byacids and enzymes in the gastro-intestinal tract; or any combinationthereof.

Further examples include, but are not limited to, the following,including synthetic, natural or recombinant sources thereof: growthhormones, including human growth hormones (hGH), recombinant humangrowth hormones (rhGH), bovine growth hormones, and porcine growthhormones; growth hormone-releasing hormones; interferons, including α,β, and γ-interferon; interleukin-1; interleukin-2; insulin, includingporcine, bovine, human, and human recombinant, optionally having counterions including sodium, zinc, calcium and ammonium; insulin-like growthfactor, including IGF-1; heparin, including unfractionated heparin,heparinoids, dermatans, chondroitins, low molecular weight heparin, verylow molecular weight heparin and ultra low molecular weight heparin;calcitonin, including salmon, eel, porcine, and human; erythropoietin;atrial naturetic factor; antigens; monoclonal antibodies; somatostatin;protease inhibitors; adrenocorticotropin, gonadotropin releasinghormone; oxytocin; leutinizing-hormone-releasing-hormone; folliclestimulating hormone; glucocerebrosidase; thrombopoietin; filgrastim;prostaglandins; cyclosporin; vasopressin; cromolyn sodium (sodium ordisodium chromoglycate); vancomycin; desferrioxamine (DFO); parathyroidhormone (PTH), including its fragments; antimicrobials, includinganti-fungal agents; vitamins; analogs, fragments, mimetics orpolyethylene glycol (PEG)-modified derivatives of these compounds; orany combination thereof. Preferred active agents include, but are notlimited to, heparin and calcitonin.

The amount of active agent in the composition is an amount effective toaccomplish the purpose intended. The amount in the composition istypically a pharmacologically, biologically, therapeutically, orchemically effective amount. However, the amount can be less than thatamount when a plurality of the compositions are to be administered,i.e., the total effective amount can be administered in cumulativeunits. The amount of active agent can also be more than apharmacologically, biologically, therapeutically, or chemicallyeffective amount when the composition provides sustained release of theactive agent. Such a composition typically has a sustained releasecoating which causes the composition to release a pharmacologically,biologically, therapeutically, or chemically effective amount of theactive agent over a prolonged period of time.

The total amount of active agent to be used can be determined by methodsknown to those skilled in the art. However, because the compositions maydeliver the active agent more efficiently than prior compositions,lesser amounts of the active agent than those used in prior dosage unitforms or delivery systems can be administered to the subject, whilestill achieving the same blood levels and/or therapeutic effects.

According to one preferred embodiment, the composition comprises adisodium salt of a delivery agent and calcitonin. Preferably, thedelivery agent is 5-CNAC. Generally, the weight ratio of calcitonin todisodium salt of 5-CNAC varies depending on the animal to which thecomposition is to be administered. For example, for a composition whichis to be administered to humans the weight ratio may range from about1:300 to about 1:700 and is preferably about 1:500. For primates, theweight ratio generally ranges from about 1:100 to about 1:500.

The composition of the present invention may be in liquid or solid form.Preferably, compositions containing the disodium salt and/or hydrate ofthe present invention are in solid form. The composition may furthercomprise additives including, but not limited to, a pH adjuster, apreservative, a flavorant, a taste-masking agent, a fragrance, ahumectant, a tonicifier, a colorant, a surfactant, a plasticizer, alubricant, a dosing vehicle, a solubilizer, an excipient, a diluent, adisintegrant, or any combination of any of the foregoing. Suitabledosing vehicles include, but are not limited to, water, phosphatebuffer, 1,2-propane diol, ethanol, olive oil, 25% aqueous propyleneglycol, and any combination of any of the foregoing. Other additivesinclude phosphate buffer salts, citric acid, glycols, and otherdispersing agents. Stabilizing additives may be incorporated into thesolution, preferably at a concentration ranging between about 0.1 and20% (w/v).

The composition may also include one or more enzyme inhibitors, such asactinonin or epiactinonin and derivatives thereof. Other enzymeinhibitors include, but are not limited to, aprotinin (Trasylol) andBowman-Birk inhibitor.

The composition of the present invention may be prepared by dry mixingor mixing in solution the disodium salt, hydrate, and/or ethanolsolvate, active agent, and, optionally, additives. The mixture may begently heated and/or inverted to aid in dispersing the components insolution.

The composition of the present invention may be formulated into a dosageunit form and in particular an oral dosage unit form, including, but notlimited to, capsules, tablets, and particles, such as powders andsachets, by methods known in the art.

According to one preferred embodiment, the dosage unit form is a soliddosage unit form comprising a lyophilized mixture of at least one of adisodium salt, ethanol solvate, or hydrate of the present invention andat least one active agent.

The term “lyophilized mixture” includes, but is not limited to, mixturesprepared in dry form by rapid freezing and dehydration. Typicallydehydration is performed while the mixture is frozen and under a vacuum.Lyophilized mixtures generally are substantially free of water andpreferably contain less than 4% by weight of water, based upon 100%total weight of the mixture.

Such a solid dosage unit form may be prepared by (a) obtaining asolution comprising one or more delivery agents and one or more activeagents, (b) lyophilizing the solution to obtain a lyophilized mixture,and (c) preparing a solid dosage unit form with the lyophilized mixture.

The delivery agent and active agent may be mixed in solution to form thesolution in step (a). The solution may be lyophilized by any methodknown in the art. The lyophilized mixture may be incorporated into adosage unit form by any method known in the art.

The composition and the dosage unit form of the present invention may beadministered to deliver an active agent to any animal in need thereofincluding, but not limited to, birds, such as chickens; mammals, such asrodents, cows, pigs, dogs, cats, primates, and particularly humans; andinsects. The composition and dosage unit form may be administered by theoral, intranasal, sublingual, intraduodenal, subcutaneous, buccal,intracolonic, rectal, vaginal, mucosal, pulmonary, transdermal,intradermal, parenteral, intravenous, intramuscular or ocular route.Preferably, the composition and dosage unit form are administeredorally.

The following examples are intended to describe the present inventionwithout limitation.

Example 1 Preparation of N-(5-chlorosalicyloyl)-8-aminocaprylic acid(5-CNAC)

To a clean, dry, 200 gallon glass-lined reactor, 178 L of dryacetonitrile was added. The agitator was set to 100-125 rpm and thereactor contents were cooled to about 9° C. 74 kg of 5-chlorosalicylamide, available from Polycarbon Industries of Leominster, Mass.,was charged to the reactor and the charging port was closed. 47 L of drypyridine was charged to the reactor. The resulting slurry was cooled toabout 9° C. Cooling was applied to the reactor condenser and valveoverheads were set for total reflux. Over 2 hours, 49.7 kg ofethylchloroformate was charged to the 200 gallon reactor whilemaintaining the batch temperature at about 14° C. Ethylchloroformate cancontain 0.1% phosgene and is extremely reactive with water. The reactionis highly exothermic and requires the use of a process chiller tomoderate reaction temperature.

The reactor contents were agitated for about 30 minutes at 10-14° C.,once the ethylchloroformate addition was complete. The reactor contentswere then heated to about 85° C. over about 25 minutes, collecting alldistillate into a receiver. The reactor contents were held at 85-94° C.for approximately 6 hours, collecting all distilled material into areceiver. The reaction mixture was sampled and the conversion (>90%)monitored by HPLC. The conversion was found to be 99.9% after 6 hours.The reactor contents were cooled to about 19° C. over a one-hour period.134 L of deionized water was charged to the reactor. A precipitateformed immediately. The reactor contents were cooled to about 5° C. andagitated for about 10.5 hours. The product continued to crystallize outof solution. The reactor slurry was centrifuged. 55 L of deionized waterwas charged to the 200-gallon, glass-lined reactor and the centrifugewet cake was washed. The intermediate was dried under full vacuum (28″Hg) at about 58° C. for about 19.5 hours. The yield was 82.6 kg6-chloro-2H-1,3-benzoxazine-2,4(3H)-dione. This intermediate waspackaged and stored so that it was not exposed to water.

In the following preparation, absolutely no water can be tolerated inthe steps up to the point where distilled water is added. 222 L of drydimethylacetamide was charged to a dry 200 gallon glass-lined reactor.The reactor agitator was set to 100-125 rpm. Cooling was applied to thecondenser and valve reactor overheads were set for distillation. 41.6 kgof dry anhydrous sodium carbonate was charged to the reactor and thereactor charging port was closed. Caution was used due to someoff-gassing and a slight exothermic reaction. 77.5 kg of dry6-chloro-2H-1,3-benzoxazine-2,4(3H)-dione was charged to the reactor.Quickly, 88 kg of dry ethyl-8-bromooctanoate was charged to the reactor.The reaction was evacuated to 22-24 inches of vacuum and the reactortemperature was raised to 65-75° C. The reactor temperature wasmaintained and the contents were watched for foaming. The reactormixture was sampled and monitored for conversion by monitoring thedisappearance of the bromo ester in the reaction mixture by gaschromatography. The reaction was complete (0.6% bromo ester was found)after about 7 hours. The vacuum was broken and the reactor contents werecooled to 45-50° C. The contents were centrifuged and the filtrate sentinto a second 200 gallon glass-lined reactor. 119 L of ethanol (200proof denatured with 0.5% toluene) was charged to the first 200 gallonreactor, warmed to about 45° C. The filter cake was washed with warmethanol and the wash was charged to the reaction mixture in the second200 gallon reactor.

The agitator was started on the second 200 gallon reactor. The reactorcontents were cooled to about 29° C. 120 L distilled water was slowlycharged to the second reactor, with the water falling directly into thebatch. The reactor contents were cooled to about 8° C. The intermediatecame out of solution and was held for about 9.5 hours. The resultantslurry was centrifuged. 70 L ethanol was charged to the reactor, cooledto about 8° C., and the centrifuge cake was washed. The wet cake wasunloaded into double polyethylene bags placed inside a paper lined drum.The yield was 123.5 kg of ethyl8-(6-chloro-2H-1,3-benzoxazine-2,4(3H)-dionyl)octanoate.

400 L purified water, USP and 45.4 kg sodium hydroxide pellets werecharged to a 200 gallon glass-lined reactor and the agitator was set to100-125 rpm. 123.5 kg of the ethyl8-(6-chloro-2H-1,3-benzoxazine-2,4(3H)-dionyl)octanoate wet cake wascharged to the reactor. The charging port was closed. Cooling water wasapplied to the condenser and the valve reactor overheads were set foratmospheric distillation. The reactor contents were heated to about 98°C. and the conversion was monitored by HLPC. Initially (approximately 40minutes) the reactor refluxed at about 68° C., however, as the ethanolwas removed (over about 3 hours) by distillation the reactor temperaturerose to about 98° C. The starting material disappeared, as determined byHPLC, at approximately 4 hours. The reactor contents were cooled toabout 27° C. 150 L purified water, USP was charged to an adjacent 200gallon glass-lined reactor and the agitator was set to 100-125 rpm. 104L concentrated (12M) hydrochloric acid was charged to the reactor andcooled to about 24° C. The saponified reaction mixture was slowlycharged (over about 5 hours) to the 200 gallon glass-lined reactor. Thematerial (45 L and 45 L) was split into 2 reactors (200 gallons each)because of carbon dioxide evolution. The product precipitated out ofsolution. The reaction mixture was adjusted to pH 2.0-4.0 with a 50%sodium hydroxide solution (2 L water, 2 kg sodium hydroxide). Thereactor contents were cooled to about 9-15° C. The intermediatecrystallized out of solution over approximately 9 hours. The reactorslurry was centrifuged to isolate the intermediate. 50 L purified water,USP was charged to a 200 gallon glass-lined reactor and this rinse wasused to wash the centrifuge wet cake. The wet cake was unloaded intodouble polyethylene bags placed inside a plastic drum. TheN-(5-chlorosalicyloyl)-8-aminocaprylic acid was dried under vacuum (27″Hg) at about 68° C. for about 38 hours. The dry cake was unloaded intodouble polyethylene bags placed inside a 55-gallon, steel unlined,open-head drums with a desiccant bag placed on top. The dried isolatedyield was 81 kg of N-(5-chlorosalicyloyl)-8-aminocaprylic acid.

Example 2 Preparation of DisodiumN-(5-chlorosalicyloyl)-8-aminocaprylate

A 22 L, Pyrex glass, five-neck, round bottom flask was equipped with anoverhead stirrer, thermocouple temperature read out, and heating mantle.The flask was charged with 2602.3 g ofN-(5-chlorosalicyloyl)-8-aminocaprylic acid and 4000 mL water. To thisstirred slurry was added a solution of 660 g of sodium hydroxidedissolved in 2000 mL water. The mixture was heated to about 55° C. andmost of the solids dissolved. The slightly hazy solution was hotfiltered through Whatman #1 filter paper to remove the insolubleparticulates. The filtrate was transferred to the pot flask of a largelaboratory rotary evaporator. The rotary evaporator was operated with abath temperature of about 60° C. and a pressure of 60 mmHg. Water wasremoved from the disodium salt solution until a solid mass was obtainedin the rotary evaporator pot flask. The vacuum was released and potflask removed from the rotary evaporator. The solids were scraped fromthe pot flask into trays. These trays were then placed in a vacuum ovenand the solids dried at about 60° C. and full vacuum for about 48 hours.The dried solids were run through a laboratory mill until all the solidspassed through a 35 mesh screen. The milled and sieved disodiumN-(5-chlorosalicyloyl)-8-aminooctanate was put into trays and placedback into the drying oven. Drying was continued at about 45° C. and fullvacuum to obtain 2957.1 g of the desired product as a dry powder.

Titration of the product with hydrochloric acid gave two inflectionpoints consuming approximately 2 molar equivalents of hydrochloric acid.CHN analysis: theoretical (correcting 4.9 wt % water) C, 47.89%; H,5.37%; N, 3.72%; Na, 12.22%; actual C, 47.69%; H, 5.23%; N, 3.45%; Na,11.79%.

Example 3 Preparation of MonosodiumN-(5-chlorosalicyloyl)-8-aminocaprylate

A 22 L, Pyrex glass, five-neck, round bottom flask was equipped with anoverhead stirrer, thermocouple temperature read out, and heating mantle.The flask was charged with 2099.7 g ofN-(5-chlorosalicyloyl)-8-aminooctanoic acid and 6000 mL water andstirred. To this slurry was added a solution of 265 g of sodiumhydroxide dissolved in 2000 mL water. The mixture was heated to about80° C. causing most of the solids to dissolve. The undissolved materialwas allowed to settle to the bottom of the flask and the supernatedecanted. The resulting mixture was transferred to the pot flask of alarge laboratory rotary evaporator. The rotary evaporator was operatedwith a bath temperature of about 60° C. and a pressure of about 70 mmHg.Water was removed from the disodium salt mixture until a solid mass wasobtained in the rotary evaporator pot flask. The vacuum was released andpot flask removed from the rotary evaporator. The solids were scrapedfrom the pot flask into trays. These trays were then placed in a vacuumoven and the solids dried at about 60° C. and full vacuum for about 48hours. The dried solids were run through a laboratory mill until all thesolids passed through a 35 mesh screen. The milled and seived disodiumN-(5-chlorosalicyloyl)-8-aminooctanate was put into trays and placedback into the drying oven. Drying was continued at full vacuum to yield2161.7 g of the desired product as a dry powder.

Titration of the product with hydrochloric acid gave a single inflectionpoint consuming approximately 1 molar equivalent of hydrochloric acid.CHN analysis: theoretical (correcting 1.14 wt % water) C, 53.05%; H,5.77%; N, 4.12%; Na, 6.77%; actual C, 52.57%; H, 5.56%; N, 4.06%; Na,6.50%.

Example 4

Disodium and monosodium salts of 5-CNAC were dosed to Rhesus monkeys asfollows. Six monkeys in one group were each dosed with one capsulecontaining the disodium salt, while six monkeys in a second group wereeach dosed with one capsule containing the monosodium salt. Each capsulewas prepared by hand-packing 400 mg 5-CNAC (mono- or di-sodium salt) and800 μg salmon calcitonin (sCT) into a hard gelatin capsule.

The Rhesus monkeys were fasted overnight prior to dosing and wererestrained in chairs, fully conscious, for the duration of the studyperiod. The capsules were administered via a gavage tube followed by 10ml water.

Blood samples were collected at 15, 30, and 45 minutes and at 1, 1.5, 2,3, 4, 5, and 6 hours after administration. Plasma concentration sCT wasdetermined by radio-immunoassay. The results from the six monkeys ineach dosing group were averaged for each time point and plotted. Themaximum mean plasma calcitonin concentration and the area under thecurve (AUC) are reported below in Table 1.

TABLE 1 Mean Peak Plasma Calcitonin Concentration (pg/ml ± StandardDelivery Agent sCT Dose Deviation) Delivery Agent Dose (mg) (mg)(Standard Error) AUC Disodium salt 400 800  424 ± 230 (94) 883 of 5-CNACMonosodium 400 800 93.2 ± 133 (54) 161 salt of 5- CNAC

Example 5

N-(10-[2-hydroxybenzoyl]amino)decanoic acid was prepared by theprocedure described in Example 1 using the appropriate startingmaterials.

Example 6 Preparation of Disodium N-salicyloyl-10-aminodecanoate EthanolSolvate

A 1 L Pyrex glass, four-neck, round bottom flask was equipped with anoverhead stirrer, reflux condenser, thermocouple temperature read out,and heating mantle. The flask was purged with dry nitrogen and thefollowing reaction conducted under an atmosphere of dry nitrogen. Theflask was charged with 100 g of N-salicyloyl-10-aminodecanoic acid and500 mL absolute ethanol. The slurry was heated to about 40° C. withstirring and all of the solids were dissolved. An addition funnel wasattached to the reactor and charged with 232.5 g of 11.2 wt % sodiumhydroxide dissolved in absolute ethanol. The sodium hydroxide solutionwas added to the stirred reaction mixture over a fifteen minute period.The reflux condenser was removed from the reactor and replaced with adistillation head and receiver. The reaction mixture was distilled atatmospheric pressure until about 395 g of distillate was collected. Thereaction mixture had become a thick slurry during this distillation. Themixture was allowed to cool to room temperature. The thick mixture wastransferred to a sintered glass funnel and the solids recovered byvacuum filtration. The ethanol wet cake was placed in a 45° C. vacuumoven and dried to constant weight at full vacuum. The dried material hada weight of about 124.6 g.

Titration of the product with hydrochloric acid gave two inflectionpoints consuming approximately 2 molar equivalents of hydrochloric acid.CHN analysis: theoretical (correcting 0.47 wt % water) C, 57.15%; H,7.37%; N, 3.51%; Na, 11.51%; actual C, 57.30%; H, 7.32%; N, 3.47%; Na,11.20%.

Example 7 Preparation of DisodiumN-(5-chlorosalicyloyl)-8-aminocaprylate Ethanol Solvate

A 12 L, Pyrex glass, four-neck, round bottom flask was equipped with anoverhead stirrer, thermocouple temperature read out, reflux condenser,and heating mantle. The flask was purged with dry nitrogen and thefollowing reaction was conducted under an atmosphere of dry nitrogen.The flask was charged with 1000 g ofN-(5-chloro-salicyloyl)-8-aminooctanic acid and 3000 mL of absoluteethanol. This slurry was heated to 55° C. with stirring to obtain aslightly hazy solution. The reactor was then charged with 2276 g of 11.2wt % sodium hydroxide dissolved in absolute ethanol as rapidly aspossible. There was a slight exothermic reaction causing the temperaturein the reactor to rise to about 64° C. and a precipitate began to form.The reflux condenser was removed and the reactor set for distillation.The reaction mixture was distilled over the next three hours to obtainabout 2566 g of distillate. The pot slurry was allowed to cool slowly toroom temperature. The product solids in the slurry were recovered byvacuum filtration through a sintered glass funnel to obtain 1390 g ofethanol wet cake. The wet cake was transferred to glass trays and placedin a vacuum oven. The cake was dried to constant weight at about 45° C.and full vacuum. The dry product had a weight of about 1094.7 g.

Titration of the product with hydrochloric acid gave two inflectionpoints consuming approximately 2 molar equivalents of hydrochloric acid.CHN analysis: theoretical (correcting 0 wt % water) C, 50.56%; H, 5.99%;N, 3.47%; Na, 11.39%; actual C, 50.24%; H, 5.74%; N, 3.50% (Na was notmeasured).

Example 8 Preparation of MonosodiumN-(10-[2-hydroxybenzoyl]amino)decanoate

A 22 L, Pyrex glass, five-neck, round bottom flask was equipped with anoverhead stirrer, thermocouple temperature read out, and heating mantle.The flask was charged with 801.8 g ofN-(10-[2-hydroxybenzoyl]amino)decanoic acid and 6000 mL water andstirred. To this slurry was added a solution of 104 g of sodiumhydroxide dissolved in 3000 mL water. The mixture was heated to about63° C. causing most of the solids to dissolve. The resulting slightlyhazy mixture was transferred to a pot flask of a large laboratory rotaryevaporator. Water was removed from the monosodium salt solution until asolid mass was obtained in the rotary evaporator pot flask. The vacuumwas released and pot flask removed from the rotary evaporator. Thesolids were scraped from the pot flask into trays. These trays were thenplaced in a vacuum oven and the solids dried at about 80° C. and fullvacuum for about 48 hours. The dried solids were identified as thedesired monosodium salt. The weight of the dried material was 822.4 g.

Titration of the product with hydrochloric acid gave one inflectionpoint consuming approximately 1 molar equivalents of hydrochloric acid.CHN analysis: theoretical (correcting 0.549 wt % water) C, 61.65%; H,7.37%; N, 4.23%; Na, 6.94%; actual C, 61.72%; H, 7.38%; N, 3.93%; Na,6.61%.

Example 9 Preparation of Disodium N-salicyloyl-10-aminodecanoate EthanolSolvate/Heparin Capsules

Disodium N-salicyloyl-10-aminodecanoate (SNAD) ethanol solvate wasscreen through a 20 mesh sieve. 7.77 g of the screened disodium SNADethanol solvate was weighed out and transferred to a mortar. 1.35 g ofheparin sodium, USP (182 units/mg), available from Scientific ProteinLaboratories, Inc., of Waunakee, Wis., was weighed out and added to thedisodium SNAD ethanol solvate in the mortar. The powders were mixed withthe aid of a spatula. The mixed powders were transferred to a 1 pintV-blender shell, available from Patterson-Kelley Co. of EastStroudsburg, Pa., and mixed for about 5 minutes.

Size 0 hard gelatin capsules, available from Torpac Inc. of Fairfield,N.J., were each hand filled with about 297-304 mg of the disodium SNADethanol solvate/heparin powder. The mean weight of the powder in eachcapsule was about 300.4 mg and the mean total weight of the capsules(i.e. the weight of the capsule with the powder) was about 387.25 g.Each capsule contained about 259.01 mg disodium SNAD ethanol solvate andabout 45.0 mg of heparin.

Example 10 Preparation of Monosodium SNAD/Heparin Tablets

Monosodium SNAD/heparin tablets were prepared as follows. SNAD wasscreened through a 35 mesh sieve. 150.3 g of SNAD, 27.33 g of heparinsodium USP (available from Scientific Protein Laboratories, Inc., ofWaunakee, Wis.), 112.43 g of Avicel™ PH 101 (available from FMACorporation of Newark, Del.), 6.0 g of Ac-Di-Sol™ (available from FMACorporation), and 2.265 g of talc (Spectrum Chemicals of New Brunswick,N.J.) were weighed out and transferred to a 2 quart V-blender shell,available from Patterson Kelley of East Stroudsburg, Pa., and blendedfor about 5 minutes. The resulting blend was compressed into slugs usingan EK-O tablet press, available from Korsch America Inc, of Sumerset,N.J. The resulting slugs were crushed and sieved through a 20 mesh sieveto produce granules. 3.94 g of talc and 5.25 g of Ac-Di-Sol were addedto the granules and transferred to a 2 quart V-blender shell and mixedfor about 5 minutes. 2.72 g of magnesium stearate were added to thegranules in the V-blender and mixed for an additional 3 minutes. Theresulting formulation was made into tablets using an EK-O tablet press.The mean tablet weight was 320.83 mg.

Example 11

4 cynomolgus macaque monkeys (2 male, 2 female) weighing about 3.0 kgeach were dosed with two of the capsules as prepared in Example 9 above.The dose for each monkey was about 150 mg/kg of the disodium SNADethanol solvate and about 30 mg/kg of heparin.

The dosing protocol for administering the capsules to each animal was asfollows. The animal was deprived food overnight prior to dosing (and 2hours post dosing). Water was available throughout the dosing period and400 ml juice was made available to the animal overnight prior to dosingand throughout the dosing period. The animal was restrained in a slingrestraint. A capsule was placed into a “pill gun”, which is a plastictool with a cocked plunger and split rubber tip to accommodate acapsule. The pill gun was inserted into the esophagus of the animal. Theplunger of the pill gun was pressed to push the capsule out of therubber tip into the esophagus. The pill gun was then retracted. Theanimal's mouth was held closed and approximately 5 ml reverse osmosiswater was administered into the mouth from the side to induce aswallowing reflex. The throat of the animal was rubbed further to inducethe swallowing reflex.

Blood samples (approximately 1.3 ml) were collected from an appropriatevein (femoral, brachial or saphenous) before dosing and 10, 20, 30, 40and 50 minutes and 1, 1.5, 2, 3, 4 and 6 hours after dosing. Bloodsamples were collected into a tube with about 0.13 ml of about 0.106 Mcitrate solution. Blood was added to fill the tube to the 1.3 ml line.The tube was then placed on wet ice pending centrifugation. Bloodsamples were centrifuged and refrigerated (2-8° C.) for about 15 minutesat 2440 rcf (approximately 3680 rpm). The resultant plasma was dividedinto 2 aliquots, stored on dry ice or frozen (at approximately −70° C.)until assayed.

Assaying

Plasma heparin concentrations were determined using the anti-Factor Xaassay CHROMOSTRATE™ heparin anti-X_(a) assay, available from OrganonTeknika Corporation of Durham, N.C. Results from the animals wereaveraged for each time point. The maximum averaged value, which wasreached at about 1 hour after administration, was 1.54±0.17 IU/mL.

Comparative Example 11A

The procedure in Example 11 was repeated with tablets of the monosodiumsalt of SNAD as prepared in Example 10 instead of the capsules of theethanol solvate of the disodium salt of SNAD. Two tablets were dosed toeach of approximately 4.0 kg monkeys. The dosage was approximately 150mg/kg SNAD (free acid equivalent) and 30 mg/kg heparin. The maximumaverage plasma heparin concentration was reached at 2 hours afteradministration and was 0.23±0.19 IU/mL.

Example 12 Preparation of Mono-SodiumN-(8-[(2-hydroxybenzoyl]amino)caprylate (SNAC) Salt

The free acid of SNAC (i.e. N-(8-[2-hydroxybenzoyl]amino)caprylic acid)was prepared by the method of Example 1 using the appropriate startingmaterials.

Into a clean 300 gallon reactor was charged 321 L of ethanol, which wasdenatured with 0.5% toluene. While stirring, 109 kg (dry) of the freeacid of SNAC was added. The reactor was heated to 28° C. and maintainedat a temperature above 25° C. A solution of 34 L purified water, USP and15.78 kg sodium hydroxide was prepared, cooled to 24° C., and added tothe stirring reactor over 15 minutes, keeping the reaction temperatureat 25-35° C. The mixture was stirred for an additional 15 minutes.

Into an adjacent reactor was charged 321 L of ethanol, which wasdenatured with 0.5% toluene. The reactor was heated to 28° C. using acirculator. The solution from the first reactor was added to the secondreactor over 30 minutes, keeping the temperature above 25° C. Thecontents were stirred and 418 L of heptane was added. The reactionmixture was cooled to 10° C., centrifuged and then washed with 60 L ofheptane. The product was collected and dried in a Stokes oven at 82° C.under 26″ Hg vacuum for about 65 hours (over a weekend). 107.5 kgmonosodium SNAC (i.e. the monosodium salt ofN-(8-[2-hydroxybenzoyl]amino)caprylic acid) was recovered.

Example 13 Preparation of SNAC Di-Sodium Salt

Free acid of SNAC (i.e. N-(8-[2-hydroxybenzoyl]amino)caprylic acid) wasprepared as follows. The monosodium SNAC prepared in Example 12 wasacidified with 1 equivalent of concentrated hydrochloric acid in waterand stirred. The solution was then vacuum filtered and vacuum dried toyield the free acid.

100 g of the free acid of SNAC was weighed into a 2 liter 4-neck roundbottomed flask and 500 ml anhydrous ethanol was added. The temperaturewas set to about 40° C. to allow the solids to go into solution. 255.7 gof 11.2% (w/w) sodium hydroxide solution in ethanol was added byaddition funnel over 15 minutes as the temperature was raised to about82° C. 383.1 g ethanol was distilled off at a head temperature of about76-79° C. over about 1.5 hours. The reaction mixture was allowed to coolto room temperature over nitrogen, held for about 2 hours, and vacuumfiltered through a coarse funnel to recover the solids. The filter cakewas washed with the filtrate, transferred to an evaporating dish, andpulled under full vacuum at room temperature overnight in a dessicator.90.5 g (68%) ethanol solvate di-sodium salt of SNAC as a pink solid wasrecovered. Melting point >200° C. (limit of instrument used). HPLC traceshowed 100 area %. NMR showed desired product. CHN forC₁₇H₂₅NO₅Na₂.0.1265H₂O) calculated: C, 54.94; H, 6.85; N, 3.77; Na,12.37. found: C, 55.04; H, 6.56; N, 3.89; Na, 12.34.

The di-sodium salt, monohydrate of SNAC was made by drying the ethanolsolvate made above at 80° C. full vacuum for 22.75 hours and cooling atroom temperature open to air to form the monhydrate. The structure ofthe hydrate was verified by elemental analysis: calculated forC₁₅H₁₉NO₄Na₂.0.127H₂O: C, 53.01; H, 6.18; N, 4.12; Na, 13.53. found: C,53.01; H, 6.10; N, 3.88; Na, 13.08. and by ¹HNMR (300 MHz, DMSO-d6): d12.35 (1H, s), 7.55 (1H, dd), 6.8 (1H, dt), 6.25 (1H, dd), 6.00 (1H,dt), 3.2 (2H, q), 1.9 (2H, t), 1.45 (4H, bq), 1.25 (6H, bm). Meltingpoint >250° C. (limit of instrument used).

Example 14 Preparation of SNAD Mono-Sodium Salt

The free acid of SNAD may be prepared by the method described in Example1 using the appropriate starting materials.

206 L ethanol denatured with 0.5% toluene and 33.87 kg SNAD were chargedto a reactor, stirred for 1 hour, and sent through a filter press. 1.7kg Celite (diatomateous earth), which is available from CeliteCorporation of Lompoc, Calif., was added to the reactor. The contents ofthe reactor were sent through a filter press and the solution wasretained in a separate vessel. The reactor was rinsed with 5 gallons ofdeionized water. The solution was reintroduced to the reactor with asodium hydroxide (NaOH) solution made from 4.5 kg NaOH in 12 L deionizedwater. The reactor contents were stirred for 30 minutes and 30 gallonsof solvent were removed by vacuum stripping at elevated temperature. Thereactor contents were cooled to 60° C. and then poured into two 100gallon tanks containing 65 gallons heptane each, with rapid stirring.Stirring was continued for 2 hours. The solution was centrifuged, washedwith 15 gallons heptane, spun dry, dried in an oven at 45° C. under 26″Hg for 24 hours, and then sent through a Fitzmill grinder (availablefrom the Fitzpatrick Company of Elmhurst, Ill.). 32 kg of the monosodiumsalt form of SNAD was recovered as a light tan powder (melting point190-192° C., 99.3% pure by HPLC, molecular weight: 329.37). Titrationrevealed about 96% mono-sodium and about 4% di-sodium salt form of SNAD.

Example 15 Preparation of SNAD Di-Sodium Salt

The free acid of SNAD (N-(10-[2-hydroxybenzoyl]amino)decanoic acid) wasprepared by the method described in Example 1 using the appropriatestarting materials.

100 g of the free acid of SNAD was weighed into a 1 liter 4-neck roundbottomed flask. 500 ml anhydrous ethanol was charged to the flask. Thetemperature was set to about 40° C. to allow the solids to go intosolution. A light orange solution was obtained. 232.5 g of 11.2 (w/w)sodium hydroxide solution in ethanol was added by addition funnel over15 minutes as the temperature was raised to about 82° C. 397.8 g ethanolwas distilled off at a head temperature of about 75-79° C. over about 3hours. The reaction mixture was allowed to cool to room temperatureovernight under nitrogen. The resulting slurry was vacuum filteredthrough a coarse funnel to recover the solids and the filter cake waswashed with the filtrate. The wet filter cake was transferred to anevaporating dish and placed into a 50° C. oven under full vacuumovernight. 124.55 g (96%) SNAD di-sodium salt, ethanol solvate as a palepink solid was recovered. Melting point >200° C. (limit of instrumentused). HPLC trace showed 100 area %. NMR showed desired product. CHN forC₁₉H₂₉NO₅Na₂) calculated: C, 57.42; H, 7.35; N, 3.52; Na, 11.57. found:C, 57.37; H, 7.35; N, 3.41; Na, 11.63.

The di-sodium salt, monohydrate of SNAD was made by drying the ethanolsolvate made above at about 80° C. full vacuum for about 19 hours andcooling the solution at room temperature open to air to form themonhydrate. The structure of the hydrate was verified by elementalanalysis: calculated for C₁₇H₂₃NO₄Na₂.H₂O: C, 55.28; H, 6.82; N, 3.79;Na, 12.45. found: C, 56.03; H, 6.67; N, 3.67; Na, 12.20. and ¹HNMR (300MHz, DMSO-d₆): d 12.35 (1H, s), 7.6 (1H, dd), 6.8 (1H, dt), 6.25 (1H,dd), 6.00 (1H, dt), 3.2 (2H, q), 2.0 (2H, t), 1.9 (2H, t), 1.45 (4H,bt), 1.25 (10H, bm). Melting point >250° C. (limit of instrument used).

Example 16 Oral Delivery of Heparin

Oral gavage (PO) dosing solutions containing heparin sodium USP andeither the mono-sodium or di-sodium salt form of the delivery agentcompound SNAC were prepared in water. The delivery agent compound andheparin (166.9 IU/mg) were mixed by vortex as dry powders. This drymixture was dissolved in water, vortexed and sonicated at about 37° C.to produce a clear solution. The pH was not adjusted. The final volumewas adjusted to about 10.0 ml. The final delivery agent compound dose,heparin dose and dose volume amounts are listed below in Table 2 below.

The typical dosing and sampling protocols were as follows. MaleSprague-Dawley rats weighing between 275-350 g were fasted for 24 hoursand were anesthetized with ketamine hydrochloride (about 88 mg/kg)intramuscularly immediately prior to dosing and again as needed tomaintain anesthesia. A dosing group of ten rats was administered one ofthe dosing solutions. An 11 cm Rusch 8 French catheter was adapted to a1 ml syringe with a pipette tip. The syringe was filled with dosingsolution by drawing the solution through the catheter, which was thenwiped dry. The catheter was placed down the esophagus leaving 1 cm oftubing past the incisors. Solution was administered by pressing thesyringe plunger.

Citrated blood samples were collected by cardiac puncture 0.25, 0.5, 1.0and 1.5 hours after administration. Heparin absorption was verified byan increase in clotting time as measured by the activated partialthromboplastin time (APTT) according to the method of Henry, J. B.,Clinical Diagnosis and Management by Laboratory Methods, Philadelphia,Pa., W.B. Saunders (1979). Previous studies indicated baseline values ofabout 20 seconds. Results from the animals in each group were averagedfor each time point and the maximum APTT value (in seconds) is reportedbelow in Table 2. Heparin absorption was also verified by an increase inplasma heparin measured by the anti-Factor Xa assay CHROMOSTRATE®Heparin anti-X_(a) assay, available from Organon Teknika Corporation ofDurham, N.C. Baseline values are about zero IU/ml. Plasma heparinconcentrations from the animals in each group were averaged for eachtime point and plotted. The peak of these mean plasma heparinconcentrations is reported below in Table 2.

TABLE 2 Mean Peak Volume Compound Heparin Mean Peak Factor Xa Com- DoseDose Dose APTT (sec) ± (IU · ml) ± pound (ml/kg) (mg/kg) (mg/kg) SE SESNAC - 3 300 100 247 ± 28.5 2.597 ± 0.13 mono SNAC - 3 300 100 300 ± 0   2.81 ± 0.17 di

Example 17 Oral Delivery of Low Molecular Weight Heparin (LMWH)

Oral dosing (PO) compositions containing low molecular weight heparin(LMWH) and either the mono-sodium or di-sodium salt form of the deliveryagent compound SNAD were prepared in water. The delivery agent compoundand LMWH (Pamaparin, 91 IU/mg, average molecular weight about 5,000),available from Opocrin of Modena, Italy, were mixed by vortex as drypowders. The dry mixture was dissolved in water, vortexed, and sonicatedat about 37° C. to produce a clear solution. The pH was not adjusted.The final volume was adjusted to about 10.0 ml. The final delivery agentcompound dose, LMWH dose, and dose volume amounts are listed below inTable 3 below.

The dosing was performed as described in Example 16 above.

Citrated blood samples were collected by cardiac puncture 0.5, 1.0, 2.0,3.0 and 4.0 hours after administration. Heparin absorption was verifiedby an increase in plasma heparin measured by the anti-Factor Xa assayCHROMOSTRATE® Heparin anti-X_(a) assay, available from Organon TeknikaCorporation of Durham, N.C. Baseline values were determined earlier andfound to be about zero IU/ml. Plasma heparin concentrations from theanimals in each group were averaged for each time point and plotted. Thepeak of these mean plasma heparin concentrations is reported below inTable 3.

TABLE 3 Mean Peak Plasma Volume Compound LMWH Heparin Com- Dose DoseDose Concentration pound (ml/kg) (mg/kg) (mg/kg) (IU/ml) ± SE SNAD- 3300 3000 0.88 ± 0.17 mono SNAD- 3 300 3000 1.21 ± 0.15 di

Example 18 Preparation of N-(5-chlorosalicyloyl)-8-aminocaprylic acid(5-CNAC)

5-chlorosalicylamide (280 g, 1.6 mol) and acetonitrile (670 ml) wereplaced in a 5 liter, 4-neck, round bottomed, flask under a nitrogenatmosphere and stirred. Pyridine (161.3 g, 2.0 mot) was added over aperiod of 25 minutes to the mixture. The reaction vessel was placed inan ice/water bath and portionwise addition of ethyl chloroformate wasstarted. This addition continued over a period of one hour. When theaddition was completed the ice/water bath was removed and the reactionmixture was allowed to come to room temperature. The reaction mixturewas allowed to stir for an additional one hour at room temperaturebefore the reaction vessel was reconfigured for distillation atatmospheric pressure. The distillation that followed yielded 257.2 g ofdistillate at a head temperature of 78° C. 500 ml of deionized water wasadded to the reaction mixture that remained in the flask and theresulting slurry was vacuum filtered. The filter cake was washed with200 ml deionized water and was allowed to dry overnight in vacuo at roomtemperature. 313.6 g (97.3%) of 6-chloro carsalam was isolated afterdrying. An additional batch was made using this same method, yielding44.5 g 6-chloro-2H-1,3-benzoxazine-2,4(3H)-dione.

Sodium carbonate (194.0 g, 1.8 mol) was added to a 5 liter, 4-neck,round bottomed, flask containing6-chloro-2H-1,3-benzoxazine-2,4(3H)-dione (323.1 g, 1.6 mol) anddimethylacetamide (970 ml). Ethyl-8-bromooctanoate (459.0 g, 1.8 mol)was added in one portion to the stirring reaction mixture. Theatmospheric pressure in the reaction vessel was reduced to 550 mm Hg andheating of the reaction mixture was started. The reaction temperaturewas maintained at 70° C. for approximately 5 hours before heating andvacuum were discontinued. The reaction mixture was allowed to cool toroom temperature overnight. The reaction mixture was vacuum filtered andthe filter cake was washed with ethyl alcohol (525 ml). Deionized water(525 ml) was slowly added to the stirred filtrate and a white solidprecipitated. An ice/water bath was placed around the reaction vesseland the slurry was cooled to 5° C. After stirring at this temperaturefor approximately 15 minutes the solids were recovered by vacuumfiltration and the filter cake was washed first with ethanol (300 ml)and then with heptane (400 ml). After drying overnight at roomtemperature in vacuo, 598.4 g (99.5%) of ethyl8-(6-chloro-2H-1,3-benzoxazine-2,4(3H)-dionyl)octanoate was obtained. Anadditional 66.6 g of ethyl8-(6-chloro-2H-1,3-benzoxazine-2,4(3H)-dionyl)octanoate was made by thissame method.

Ethyl 8-(6-chloro-2H-1,3-benzoxazine-2,4(3H)-dionyl)octanoate (641 g,1.7 mol) and ethyl alcohol (3200 ml) were added to a 22 liter, five neckflask. In a separate 5 liter flask, sodium hydroxide (NaOH) (288.5 g,7.2 mol) was dissolved in deionized water (3850 ml). This mixture wasadded to the reaction mixture contained in the 22 liter flask. Atemperature increase to 40° C. was noted. Heating of the reactionmixture was started and when the reaction temperature had increased to50° C. it was noted that all of the solids in the reaction mixture haddissolved. A temperature of 50° C. was maintained in the reactionmixture for a period of 1.5 hours. The reaction flask was then set upfor vacuum distillation. 2200 ml of distillate were collected at a vaportemperature of 55° C. (10 mm Hg) before the distillation wasdiscontinued. The reaction flask was then placed in an ice/water bathand concentrated hydrochloric acid (HCl) (752 ml) was added over aperiod of 45 minutes. During this addition the reaction mixture wasnoted to have thickened somewhat and an additional 4 liters of deionizedwater was added to aid the stirring of the reaction mixture. Thereaction mixture was then vacuum filtered and the filter cake was washedwith 3 liters of deionized water. After drying in vacuo at roomtemperature 456.7 g (83.5%) of N-(5-chlorosalicyloyl)-8-aminocaprylicacid was isolated.

Example 19 Lyophilization of Salmon Calcitonin (sCT) and the Sodium Saltof 5-CNAC Preparation of the Sodium Salt of 5-CNAC

The percent purity of 5-CNAC was determined as follows. 0.9964 g of thefree acid of 5-CNAC was quantitatively dissolved in 40 ml of methanol. 2ml of distilled water was added to this solution after the solids weredissolved. The solution was titrated in methanol with 0.33 N sodiumhydroxide using a computer controlled burette (Hamilton automaticburette available from Hamilton of Reno, Nev.). A glass electrode(computer controlled Orion model 525A pH meter available from VWRScientific of South Plainfield, N.J.) was used to monitor the pH of thesolution. The solution was stirred with a magnetic stirrer.

The volume of titrant to reach the second pH inflection point was 18.80ml. The inflection point, determined by interpolation between the twodata points where the second derivative of the pH plot changed frompositive to negative, occurred at pH 11.3. The purity of the free acidwas determined using the following equation:

${\% \mspace{14mu} {purity}} = \frac{\begin{matrix}{100 \times \left( {{Volume}\mspace{14mu} {of}\mspace{14mu} {Titrant}\mspace{14mu} {in}\mspace{14mu} {ml}} \right) \times} \\{{Normality} \times {Molecular}\mspace{14mu} {weight}}\end{matrix}}{1000 \times {Equivalents} \times {Sample}\mspace{14mu} {Weight}}$

where Normality is the normality of sodium hydroxide, Molecular Weightis the molecular weight of 5-CNAC free acid (313.78), Equivalents is theequivalence of free acid (2 in this case, since it is dibasic), andSample Weight is the weight of the free acid sample being titrated.

The purity was found to be 97.0%.

9.3458 g 5-CNAC powder was weighed out. The amount of 0.33 N sodiumhydroxide needed to have a sodium hydroxide to free acid molar ratio of1.6 was calculated using the following equation:

${{Volume}\mspace{14mu} {of}\mspace{14mu} {NaOH}\mspace{14mu} \left( {{in}\mspace{14mu} {ml}} \right)} = \frac{{Free}\mspace{14mu} {Acid}\mspace{14mu} {Weight} \times \left( {\% \mspace{14mu} {purity}} \right) \times 1000 \times 1.6}{313.78 \times 100 \times {Normality}}$

where the Free Acid Weight is the weight of free acid in formulatedsample, the % purity is the percentage purity of 5-CNAC, Normality isthe normality of sodium hydroxide, and the Volume of NaoH is the amountof sodium hydroxide needed.

5-CNAC and 153.3 ml of 0.33 N sodium hydroxide (NaOH) was mixed in aPyrex bottle. The resulting slurry was warmed in a steam bath to 60-80°C. The warm slurry became a clear solution in about 15 minutes withoccasional stirring. The solution was cooled to room temperature. The pHof this solution was 8.1.

Preparation of sCT/Sodium Salt of 5-CNAC Solution

The aqueous solution of 5-CNAC sodium salt was filtered through asterile, 0.45 micron cellulose acetate, low protein binding membrane ona 150 ml Corning filter (available from VWR Scientific Product, S.Plainfield, N.J.). The pH of the solution was about 8.3.

Dry salmon calcitonin (sCT), stored at −70° C., was brought to roomtemperature. Next, 18.692 mg of sCT was weighed out and dissolved in 10ml of 0.1 M mono sodium phosphate buffer solution at a pH of about 5,with gentle mixing.

The sCT solution was added to the 5-CNAC sodium salt solution withgentle mixing, taking precaution to avoid foaming or vortexing.

Lyophilization of sCT/Sodium Salt of 5-CNAC Solution

Shelves of the lyophilizer (Genesis 25 LL-800 from The Virtis Company ofGardiner, N.Y.) were prefrozen to about −45° C.

Approximately 260 ml of sCT/sodium salt of 5-CNAC solution was added toa 30 cm×18 cm stainless steel tray to give a cake thickness of about0.48 cm. Four clean, dry thermocouple probe tips were inserted into thesolution such that the probe tip touched the solution level in thecenter. The probes were secured with clips to the side of the tray andthe trays were loaded on to the precooled shelves.

The gel permation chromatograph (GPC2) was programmed for the cycleshown in Table 4.

TABLE 4 Lyophilization Process Cycle Step Temperature Pressure set point(m torr) Time (minute) 1 −45° C. none (Prefreeze) 120 2 −30° C. 300 1803 −20° C. 200 200 4 −10° C. 200 360 5  −0° C. 200 720 6  10° C. 100 5407  20° C. 100 360 8  25° C. 100 180

During lyophilization the pressure varied from 350 to 45 mtorr. When thelyophilization cycle was completed, the system cycle was terminated andthe system vacuum was released. The trays were carefully removed fromthe shelves and the lyophilized powder was transferred into amber HDPENALGENE® bottles, available from VWR Scientific.

Using the above cycle for lyophilization, a powder with about 3%moisture content was obtained. The powder was hand packed into hardgelatin capsules (size OEL/CS), which are available from Capsugel, adivision of Warner Lamber Co., of Greenwood, S.C., as needed. The filledcapsules and the lyophilized powder were stored in a closed containerwith dessicant.

Example 20 Preparation of Unlyophilized sCT/Sodium Salt of 5-CNAC

Acetic anhydride (56.81 ml, 61.47 g, 0.6026 mol), 5-chlorosalicylic acid(100.00 g, 0.5794 mol), and xylenes (200 ml) were added to a 500 ml,three-neck flask fitted with a magnetic stir bar, a thermometer, and aDean-Stark trap with condenser. The flask was heated to reflux, thereaction mixture clearing to a yellow solution around 100° C. Most ofthe volatile organics (xylenes and acetic acid) were distilled into theDean-Stark trap (135-146° C.). Distillation was continued for anotherhour, during which the pot temperature slowly rose to 190° C. and thedistillate slowed to a trickle to drive over any more solvent.Approximately 250 ml of solvent was collected. The residue was cooledbelow 100° C. and dioxane was added.

A 2N sodium hydroxide (222.85 ml, 0.4457 mol) and 8-aminocaprylic acid(70.96 g, 0.4457 mol) solution was added to the solution ofoligo(5-chloroasalicylic acid) (0.5794 mol) in dioxane. The reactionmixture was heated to 90° C. for 5.5 hours, then shut off overnight andrestarted in the morning to heat to reflux (after restarting the heatingthe reaction was monitored at which time the reaction was determined tohave finished, by HPLC). The reaction mixture was cooled to 40° C. Thedioxane was stripped off in vacuo. The residue was taken up in 2N sodiumhydroxide and acidified. The material did not solidify. The material wasthen taken up in ethyl acetate and extracted (2×100 ml) to remove excessdioxane. The ethyl acetate layer was dried over sodium sulfate andconcentrated in vacuo. The easily filtered solids were collected byfiltration. The remaining material was taken up in 2N NaOH. The pH wasadjusted to 4.3 to selectively isolate product from starting material.Once at pH 4.3, the solids were filtered off and recrystallized in a 1:1mixture of ethanol and water. Any insoluble material was hot filteredout first. All the solids which were collected were combined andrecrystallized from the mixture of ethanol and water to give 52.06 g ofthe free acid product as a white solid.

The sodium salt solution was prepared according to the method describedin Example 19 using 0.2 N NaOH solution. Percent purity was calculatedto be 100% using 0.5038 g of 5-CNAC and 16.06 ml of 0.2 N NaOH. Thesodium salt solution was prepared using 250 ml of 0.2 N NaOH and 9.4585g of 5-CNAC prepared as described above. The solution was filteredthrough a 0.45 micron filter.

Example 21 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats

Male Sprague-Dawley rats weighing between 200-250 g were fasted for 24hours and were administered ketamine (44 mg/kg) and chlorpromazine (1.5mg/kg) 15 minutes prior to dosing. The rats were administered one of thefollowing:

(4a) orally, one capsule of 13 mg lyophilized powder as prepared as inExample 19 with 0.5 ml of water to flush the capsule down;

(4b) orally, 1.0 ml/kg of a reconstituted aqueous solution of thelyophilized powder prepared in Example 19;

(4c) orally, 1.0 ml/kg of “fresh”, unlyophilized aqueous solution of5-CNAC sodium salt as prepared in Example 20 with sCT; or

(4d) subcutaneously, 5 mg/kg of sCT.

Doses (4a), (4b) and (4c) contained 50 mg/kg of the sodium salt of5-CNAC and 100 mg/kg of sCT. Doses for (4a) are approximate because theanimals were given one capsule filled with the stated amount of powderbased on an average animal weight of 250 g, whereas actual animal weightvaried. This is also the case in all later examples where a capsule isdosed.

The reconstituted solution for (4b) was prepared by mixing 150 mg of thelyophilized powder prepared as in Example 19 in 3 ml of water. Thereconstituted solution was dosed at 1.0 ml/mg.

The “fresh” solution for (4c) was prepared from unlyophilized materialusing 150 mg 5-CNAC sodium salt prepared in Example 20 in 3 ml waterplus 150 ml of sCT stock solution (2000 ml/ml prepared in 0.1M phospatebuffer, pH adjusted to 4 with HCl and NaOH. The “fresh” solution had afinal concentration of 50 mg/ml 5-CNAC sodium salt and of 100 mg/ml sCT,and 1.0 ml/kg was dosed.

The subcutaneous doses were prepared by dissolving 2 mg of sCT in 1 mlwater. 5 mL of this solution was added to 995 mL of water. This solutionwas dosed at 0.5 ml/kg.

Blood samples were collected serially from the tail artery. Serum sCTwas determined by testing with an EIA kit (Kit # EIAS-6003 fromPeninsula Laboratories, Inc., San Carlos, Calif.), modifying thestandard protocol from the kit as follows: incubated with 50 ml peptideantibody for 2 hours with shaking in the dark, washed the plate, addedserum and biotinylated peptide and diluted with 4 ml buffer, and shookovernight in the dark. Results are illustrated in Table 5, below.

TABLE 5 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats Dose ofSodium Mean Peak Salt of 5-CNAC sCT Dose Serum sCT ± SD Dosage form(mg/kg) (mg/kg) (pg/ml) (4a) capsule  50*  100* 1449 ± 2307 (4b)reconstituted 50 100 257 ± 326 solution (4c) unlyophilized 50 100 134 ±169 solution (4d) subcutaneous —  5 965 ± 848 *approximate dose due tovariations in animal weight

Example 22 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats

According to the method described in Example 21, rats were administeredone of the following:

(5a) orally, one capsule of 13 mg lyophilized powder with 1 ml water toflush the capsule down;

(5b) orally, one capsule of 6.5 mg lyophilized powder with 1 ml water toflush the capsule down;

(5c) orally, one capsule of 3.25 mg lyophilized powder with 1 ml waterto flush the capsule down;

(5d) subcutaneously 5 mg/kg of sCT.

Approximate amounts of delivery agent and sCT, as well as the results,are shown in Table 6 below.

TABLE 6 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats Dose ofSodium Mean Peak Salt of 5-CNAC sCT Dose Serum sCT ± SD Dosage form(mg/kg) (mg/kg) (pg/ml) (5a) capsule 50* 100*  379 ± 456 (5b) capsule25* 50* 168 ± 241 (5c) capsule   12.5* 25* 0 (5d) subcutaneous — 5 273 ±320 *approximate dose due to variations in animal weight

Example 23 Preparation of N-(5-chlorosalicyloyl)-4 aminobutyric acid

Sodium carbonate (30 g, 0.2835 mol) was added to a 500 ml 3-neck,round-bottomed flask containing6-chloro-2H-1,3-benzoxazine-2,4(3H)-dione (prepared as in Example 18)(50 g, 0.2532 mol) and dimethylacetamide (75 ml) and stirred.Methyl-4-bromobutyrate (45.83 g, 0.2532 mol) was added in one portion tothe stirring reaction mixture, and heating of the reaction mixture wasstarted. The reaction temperature was maintained at 70° C. and allowedto heat overnight. Heating was discontinued, and the reaction mixturewas allowed to cool to room temperature.

The reaction mixture was vacuum filtered and the filter cake was washedwith ethyl alcohol. The filter cake and filtrate were monitored by HPLCto determine where the product was. Most of the product was washed intothe filtrate, although some product was still present in the filtercake. The filter cake was worked up to recover product to increase thefinal yield. The filter cake was washed first with copious amounts ofwater, then with ethyl acetate. The washes from the filter cake wereseparated and the ethyl acetate layer was next washed twice with water,once with brine, then dried over sodium sulfate, isolated andconcentrated in vacuo to recover more solids (solids B). Water was addedto the filtrate that had been isolated earlier and solids precipitatedout. Those solids were isolated (solids A). Solids A and B were combinedand transferred to a round bottom flask and 2N NaOH was added to thefiltrate and heating was begun with stirring. The reaction was monitoredby HPLC to determine when the reaction was done. The reaction was cooledto 25° C., stirred overnight, and concentrated in vacuo to remove excessethanol. An ice/water bath was placed around the reaction vessel and theslurry was acidified. The solids were recovered by vacuum filtration andthe filter cake was washed with water, dried and sent for NMR analysis.

The solids were isolated and transferred to an Erlenmeyer flask to berecrystallized. The solids were recrystallized with methanol/water.Solids formed and were washed into a Buchner funnel. More solidsprecipitated out in the filtrate and were recovered. The first solidsrecovered after recrystallization had formed a methyl ester. All thesolids were combined, 2N NaOH was added and heated again to reflux toregain the free acid. Once the ester had disappeared, as determined byHPLC, acidification of the mixture to a pH of about 4.7 caused solids todevelop.

The solids were isolated by filtration and combined with all the solidsand recrystallized using a 1.5:1.0 ratio of methanol to water. Whitesolids precipitated out overnight and were isolated and dried to give23.48 g of N-(5-chlorosalicyloyl)-4 aminobutyric acid at a 36% yield.

It was later determined that the filter cake should have first beenwashed with excess ethyl alcohol to avoid having the product remain inthe filter cake. From that point, the filtrate and 2N NaOH could beheated with stirring, cooled to 25° C. and concentrated in vacuo toremove excess ethanol. In an ice/water bath, the slurry acidified to apH of 4.7. The solids recovered by vacuum filtration and the filter cakewere washed with water. The solids were then isolated andrecrystallized.

Example 24 Lyophilization of sCT/Sodium Salt of N-(5-chlorosalicyloyl)-4aminobutyric Acid

Following the procedure in Example 19, a lyophilized powder ofsCT/sodium salt of N-(5-chlorosalicyloyl)-4 aminobutyric acid wasprepared and packed into capsules. 10.528 g of N-(5-chlorosalicyloyl)-4aminobutyric acid as prepared in Example 23 was dissolved in 150 mlwater. 4.72 ml 10N NaOH was added. 21.0566 mg of sCT was dissolved in 10ml phosphate buffer and the sCT/phosphate buffer mixture was added tothe delivery agent solution. Water was added to make the volume 250 ml.

Example 25 Oral Delivery of sCT/Sodium Salt of N-(5-chlorosalicyloyl)-4Aminobutyric Acid in Rats

According to the method of Example 21, with the exception that thestandard protocol for the EIA kit was followed, rats were administeredorally one capsule of 13 mg lyophilized powder with 0.5 ml water toflush the capsule down with the approximate amounts of the sodium saltof N-(5-chlorosalicyloyl)-4 aminobutyric acid and sCT as set forth inTable 7 below. The results are also shown in Table 7.

TABLE 7 Oral Delivery of sCT/Sodium Salt of N-(5-chlorosalicyloyl)-4aminobutyric acid in Rats Dose of Sodium Salt of N-(5-chlorosalicyloyl)-4 Mean Peak aminobutyric acid sCT Dose Serum sCT ± SDDosage form (mg/kg) (mg/kg) (pg/ml) (8a) capsule 50* 400* 1112 ± 1398(8b) capsule 50* 800* 2199 ± 4616 *approximate dose due to variations inanimal weight

Example 26 Preparation of 5-CNAC for Tableting

To a clean, dry, 200 gallon glass-lined reactor, 178 L of dryacetonitrile was added. The agitator was set to 100-125 RPM and thereactor contents were cooled to 9° C. 74 kg of 5-chloro salicylamide,available from Polycarbon Industries of Leominster, Mass., was chargedto the reactor and the charging port was closed. 47 L of dry pyridinewas charged to the reactor. The slurry was cooled to 9° C. prior toproceeding. Cooling was applied to the reactor condenser and valveoverheads were set for total reflux. Over 2 hours, 49.7 kg ofethylchloroformate was charged to the 200 gallon reactor whilemaintaining the batch temperature at 14° C. Note that ethylchloroformatecan contain 0.1% phosgene and is extremely reactive with water. Thereacton is highly exothermic and requires the use of a process chillerto moderate reaction temperature. The reactor contents were agitated for30 minutes at 10-14° C. once the ethylchloroformate addition wascomplete. The reactor contents were heated to 85° C. over 25 minutes,collecting all distillate into a receiver. The reactor contents wereheld at 85-94° C. for approximately 6 hours, collecting all distilledmaterial into a receiver. The reaction mixture was sampled and theconversion (>90%) monitored by HPLC. The conversion was found to be99.9% after 6 hours. The reactor contents were cooled to 19° C. over aone-hour period. 134 L of deionized water was charged to the reactor. Aprecipitate formed immediately. The reactor contents were cooled to 5°C. and agitated for 10.5 hours. The product continued to crystallize outof solution. The reactor slurry was centrifuged. 55 L of deionized waterwas charged to the 200-gallon, glass-lined reactor and the centrifugewet cake was washed. The intermediate was dried under full vacuum (28″Hg) and 58° C. for 19.5 hours. The yield was 82.6 kg6-chloro-2H-1,3-benzoxazine-2,4(3H)-dione. This intermediate waspackaged and stored so that it was not exposed to water.

In the next preparation, absolutely no water can be tolerated in thesteps up to the point where distilled water is added.

222 L of dry dimethylacetamide was charged to a dry 200 gallonglass-lined reactor. The reactor agitator was set to 100-125 RPM.Cooling was applied to the condenser and valve reactor overheads wereset for distillation. 41.6 kg of dry anhydrous sodium carbonate wascharged to the reactor and the reactor charging port was closed. Cautionwas used due to some off-gassing and a slight exotherm. 77.5 kg of dry6-chloro-2H-1,3-benzoxazine-2,4(3H)-dione was charged to the reactor.Quickly, 88 kg of dry ethyl-8-bromooctanoate was charged to the reactor.22-24 inches of vacuum was applied and the reactor temperature wasraised to 65-75° C. The reactor temperature was maintained and thecontents were watched for foaming. The reactor mixture was sampled andmonitored for conversion by monitoring for the disappearance of thebromo ester in the reaction mixture by gas chromatography (GC). Thereaction was complete (0.6% bromo ester was found) after 7 hours. Thevacuum was broken and the reactor contents cooled to 45-50° C. Thecontents were centrifuged and the filtrate sent into a second 200-gallonglass-lined reactor. 119 L of ethanol (200 proof denatured with 0.5%toluene) was charged to the first 200-gallon reactor, warmed to 45° C.and the filter cake washed with warm ethanol, adding to the reactionmixture in the second 200-gallon reactor. The agitator was started onthe second 200-gallon reactor. The reactor contents were cooled to 29°C. 120 L of distilled water was slowly charged to the second reactor,with the water falling directly into the batch. The reactor contentswere cooled to 8° C. The intermediate came out of solution and was heldfor 9.5 hours. The resultant slurry was centrifuged. 70 L of ethanol wascharged to the reactor, cooled to 8° C. and the centrifuge cake waswashed. The wet cake was unloaded into double polyethylene bags placedinside a paper lined drum. The yield was 123.5 kg of ethyl8-(6-chloro-2H-1,3-benzoxazine-2,4(3H)-dionyl)octanoate.

400 L of purified water, USP and 45.4 kg NaOH pellets were charged to a200 gallon glass-lined reactor and the agitator was set to 100-125 RPM.123.5 kg of the ethyl8-(6-chloro-2H-1,3-benzoxazine-2,4(3H)-dionyl)octanoate wet cake wascharged to the reactor. The charging port was closed. Cooling waterapplied to the condenser and the valve reactor overheads were set foratmospheric distillation. The reactor contents were heated to 98° C. andconversion monitored by HPLC. Initially (approximately 40 minutes) thereactor refluxed at 68° C., however, as the ethanol was removed (over 3hours) by distillation the reactor temperature rose to 98° C. Thestarting material disappeared, as determined by HPLC, at approximately 4hours. The reactor contents were cooled to 27° C. 150 L of purifiedwater and USP were charged to an adjacent 200 gallon glass-lined reactorand the agitator was set to 100-125 RPM. 104 L of concentrated (12M)hydrochloric acid was charged to the reactor and cooled to 24° C. Thesaponified reaction mixture was slowly (over 5 hours) charged to the200-gallon glass-lined reactor. The material (45 L and 45 L) was splitinto 2 reactors (200 gallons each) because of carbon dioxide evolution.The product precipitated out of solution. The reaction mixture wasadjusted to a pH of 2.0-4.0 with 50% NaOH solution (2 L water, 2 kgNaOH). The reactor contents were cooled to 9-15° C. The intermediatecrystallized out of solution over approximately 9 hours. The reactorslurry was centrifuged to isolate the intermediate. 50 L of purifiedwater and USP were charged to a 200-gallon glass-lined reactor and thisrinse was used to wash the centrifuge wet cake. The wet cake wasunloaded into double polyethylene bags placed inside a plastic drum. TheN-(5-chlorosalicyloyl)-8-aminocaprylic acid was dried under vacuum (27″Hg) at 68° C. for 38 hours. The dry cake was unloaded into doublepolyethylene bags placed inside a 55-gallon, steel unlined, open-headdrums with a desiccant bag placed on top. The dried isolated yield was81 kg of N-(5-chlorosalicyloyl)-8-aminocaprylic acid.

Example 27 Lyophilization of sCT/Sodium Salt of 5-CNAC for Tableting

The method of Example 19 was used to prepare lyophilized powder using200 g of 5-CNAC as prepared in Example 26. The NaOH solution was made bydissolving 42 g of 100% NaOH into 2000 ml water. The slurry was stirredat room temperature, and vacuum filtered over a 0.45 micron filter. ThepH of the solution containing the sodium salt of 5-CNAC was about 8.6.200 mg of sCT was used.

Example 28 Preparation of sCT/Sodium Salt of 5-CNAC Tablets

Tablets of the lyophilized powder prepared in Example 27 were preparedas follows.

An instrumented Carver press (Model C), available from Carver of Wabash,Ind., was used for tablet compression. The die used was 0.245″ indiameter. The top punch was flat-faced, bevel-edged and 0.245″ indiameter while the bottom punch was flat-faced, scored, bevel-edged and0.245″ in diameter. The press was capable of measuring the upper andlower punch force as well as the displacement of the upper punch. Aformula for direct compression was designed as shown in Table 8 below:

TABLE 8 Material mg/tablet mg/300 tablet batch Lyophilized powder 100.230,060.0 of sCT/sodium salt of 5-CNAC AC-DI-SOL ® 2.004 601.2 MagnesiumStearate 0.511 153.3 CAB-O-SIL ® 0.205 61.5 Total Weight (mg) 102.9230,876.0 AC-DI-SOL ® is croscarmellose sodium (NF, PH. Eur., JPE) and isavailable from FMC Corporation, Pharmaceutical Division, ofPhiladelphia, PA. CAB-O-SIL ® is fumed silica and is available fromCabot Corporation, Tuscola, IL.

The Ac-Di-Sol® and Cab-O-Sil® were weighed and transferred to a mixingbottle. The bottle was then closed and secured to the arm of a sustainedrelease apparatus set at 25 rotations per, minute (RPM). The apparatuswas rotated for 5 minutes to mix. The lyophilized powder of 5-CNAC/sCTwas then added to the AC-DI-SOL/CAB-O-SIL® mixture geometrically with atwo minute mixing cycle after each addition. Magnesium stearate was thenadded to the above mixture and mixing was continued for five minutes.

Approximately 103 mg of the above powder was then transferred to the diecontaining the lower punch. The powder was pressed down into the dieusing the upper punch. The upper punch was inserted and the punch dieassembly was mounted onto the press. Compression was then performed. Theupper punch was used to push the tablet out of the die.

Example 29 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats Tablets

The tablets prepared in Example 28 were pulverized and hand packed intocapsules at 13 mg/capsule. Untableted, lyophilized powder as prepared inExample 27 was hand packed into capsules at 13 mg/capsule. The capsuleswere dosed with 1 ml water to flush them down.

Following the procedure of Example 21, with the exception that thestandard protocol for the ETA kit was followed instead of the modifiedversion, rats were administered orally one capsule with 1 ml of water toflush the capsule down with the approximate amounts of sodium salt of5-CNAC and sCT as set forth in Table 9 below. The results are also shownin Table 9.

TABLE 9 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats Dose ofSodium Mean Peak Salt of 5-CNAC sCT Dose Serum sCT ± SD Dosage form(mg/kg) (mg/kg) (pg/ml) (12a) tableted powder 50* 100* 198 ± 132 incapsule (12b) untableted 50* 100* 197 ± 125 powder in capsule*approximate dose due to variations in animal weight

Example 30 Preparation of 5-CNAC

5-CNAC was made under similar conditions as in Example 26 in alaboratory environment.

Example 31 Lyophilization of sCT/Sodium Salt of 5-CNAC

5-CNAC as prepared in Example 30 was formulated into a lyophilizedpowder with sCT as in Example 19 with 485 ml 0.2 N NaOH and 19.0072 g of5-CNAC in a steam bath. The final volume was 505 ml. Four separatebatches were prepared from 187, 138, 74 and 160 ml of the sodium salt5-CNAC with 28, 48, 40 and 360 mg sCT, respectively. The estimatedamounts of the sodium salt of 5-CNAC were 7, 5, 2.5 and 4.5 g,respectively.

Example 32 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats

According to the method of Example 21, with the exception that thestandard protocol for the EIA kit was followed instead of the modifiedversion, rats were administered orally one capsule of 13 mg lyophilizedpowder using one of the four batches prepared in Example 31, with 1 mlwater to flush the capsule down. The approximate amounts of the sodiumsalt of 5-CNAC and sCT are set forth in Table 10 below. The results areshown in Table 10.

TABLE 10 Oral Delivery of sCT/Sodium Salt of 5-CNAC in Rats Dose ofSodium Mean Peak Salt of 5-CNAC sCT Dose Serum sCT ± SD Dosage form(mg/kg) (mg/kg) (pg/ml) (15a) capsule 50* 100* 125 ± 153 (15b) capsule50* 400* 178 ± 354 (15c) capsule 50* 800* 546 ± 586 (15d) capsule 50*4000*   757 ± 1234 *approximate dose due to variations in animal weight

All patents, publications, applications, and test methods mentionedabove are hereby incorporated by reference. Many variations of thepresent matter will suggest themselves to those skilled in the art inlight of the above detailed description. All such obvious variations arewithin the patented scope of the appended claims.

1-28. (canceled)
 29. An oral dosage form comprising the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid and calcitonin.
 30. The oral dosage form of claim 29, wherein the calcitonin is salmon calcitonin.
 31. The oral dosage form of claim 29, wherein the dosage form is in solid form.
 32. The oral dosage form of claim 29, wherein the dosage form is a tablet.
 33. The oral dosage form of claim 29, wherein the dosage form is a capsule.
 34. The oral dosage form of claim 29, wherein the dosage from comprises from about 50% to about 100% by weight of the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, based upon 100% total weight of N-(5-chlorosalicyloyl)-aminocaprylic acid and salts thereof in the dosage form.
 35. The oral dosage form of claim 29, wherein the dosage from comprises from about 50% to about 90% by weight of the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, based upon 100% total weight of N-(5-chlorosalicyloyl)-aminocaprylic acid and salts thereof in the dosage form.
 36. The oral dosage form of claim 29, wherein the dosage from comprises from about 50% to about 96% by weight of the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, based upon 100% total weight of N-(5-chlorosalicyloyl)-aminocaprylic acid and salts thereof in the dosage form.
 37. The oral dosage form of claim 29, wherein the dosage from comprises at least about 90% by weight of the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, based upon 100% total weight of N-(5-chlorosalicyloyl)-aminocaprylic acid and salts thereof in the dosage form.
 38. The oral dosage form of claim 29, wherein the dosage from comprises at least about 96% by weight of the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid, based upon 100% total weight of N-(5-chlorosalicyloyl)-aminocaprylic acid and salts thereof in the dosage form.
 39. The oral dosage form of claim 29, wherein the disodium salt is in a monohydrate form.
 40. The oral dosage form of claim 29, wherein the disodium salt is anhydrous.
 41. The oral dosage form of claim 29, wherein the dosage form further comprises an excipient, a diluent, a disintegrant, a lubricant, a plasticizer, a colorant, a dosing vehicle, or any combination thereof.
 42. The oral dosage form of claim 29, wherein the weight ratio of calcitonin to the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is from about 1:300 to about 1:700.
 43. The oral dosage form of claim 29, wherein the weight ratio of calcitonin to the disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid is from about 1:100 to about 1:500.
 44. The oral dosage form of claim 29, wherein the dosage form comprises a lyophilized mixture of a disodium salt of N-(5-chlorosalicyloyl)-8-aminocaprylic acid and calcitonin.
 45. The oral dosage form of claim 44, wherein the lyophilized mixture is substantially free of water. 